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Screening method for in urine by UHPLC-MS/MS. Identification of isomeric compounds

Núria Monforta, Laura Martíneza, Rosa Bergésa, Jordi Seguraa,b, Rosa Venturaa,b a) Grup de Recerca en Bioanàlisi i Serveis Analítics, IMIM-Hospital del Mar, Barcelona, Spain b) Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, UPF, Barcelona, Spain

Corresponding author: Rosa Ventura Grup de Recerca en Bioanàlisi i Serveis Analítics IMIM (Institut Hospital del Mar d’Investigacions Mèdiques) Dr. Aiguader, 88 08003 Barcelona, Spain Tel. 34 93 3160471 Fax 34 93 3160499 Email: [email protected]

Keywords : stimulants, UHPLC-MS/MS, isomeric compounds ABSTRACT

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultraperformance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 μm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the MRPL required by WADA and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented. INTRODUCTION

The list of prohibited substances and methods of the World Antidoping Agency (WADA) includes more than two hundred pharmacologically and chemically different substances and prohibited methods[1]. Doping control laboratories need to monitor the prohibited substances and/or their metabolites mainly in urine samples. In case of positive results during the initial testing procedure (screening), laboratories apply a second analysis for confirmation purposes. Screening and confirmation analyses have to be performed in a short period of time according to the International Standard for Laboratories[2], specially important during major sports events where 24 h reporting times are needed. Therefore, laboratories are forced to develop high throughput screening and confirmation methods. The use of fast methods has been also a demand in clinical testing for a long time; diagnostic is facilitated by fast analysis, which is important in some emergency cases.

Doping control laboratories use multi-analyte methods to screen for groups of prohibited substances[3]. For long time, screening and confirmation methods were based on gas chromatography coupled to mass spectrometry (GC-MS)[4]. However, at present, liquid chromatography coupled to mass spectrometry (LC- MS) is currently the technique of choice for the screening and confirmation of most of the groups of substances of doping interest[5-24].

Different strategies have been described in order to reduce the complexity of the doping control analyses and the total analysis time. LC-MS, either tandem MS or high resolution MS, allow the comprehensive screening of different groups of substances with a simple sample preparation without the need of specific derivatizations as it was needed for GC-MS based methods[4]. In recent years, the availability of robust, sensitive and reliable MS detectors allows the development of methods where sample preparation and/or purification steps are not needed (direct injection or “dilute and shoot” methods)[18,19,22,24-26]. The direct analysis of conjugated metabolites by LC-MS has also been described to avoid the hydrolysis step during sample preparation[27]. “Dilute and shoot” methods are also especially interesting for compounds with high volatility (e.g. some compounds of the group of stimulants) that may be lost during the evaporation step of the organic solvent used in liquid-liquid or solid-phase extractions[28].

Another strategy to reduce the analysis time is the use of ultraperformance liquid chromatography (UHPLC) that enables the work with columns packed with small particles (< 2µm). UHPLC is able to produced high resolution by using high linear velocities which results in a reduction of the analysis time. Methods using UHPLC coupled to tandem MS or to high resolution MS have already been described for the screening and confirmation of some doping agents[15,18-20,23,26], demonstrating the reliability of that approach for screening purposes.

Stimulants are a group of substances nowadays frequently analyzed in doping control and analytical toxicology by “dilute and shoot” techniques[18,19,22,24]. They contain an easily-ionizable nitrogen atom and, therefore, the ionization efficiency using electrospray ionization is high. Moreover, although they are metabolized, most of them are excreted in urine in unchanged form in considerable amounts and the detection level required is high[29]. All these factors make “dilute-and- shoot” techniques a good choice for screening analysis of stimulants.

The objective of the work was to develop a fast method to screen for stimulants. A “dilute and shoot” method able to detect more than 60 stimulants by UHPLC- MS/MS is described. Due to isomeric nature of most stimulants, the possibilities for unequivocal identification of the sets of various isomers are also presented. EXPERIMENTAL

Chemicals and reagents

Standards used were: Levmetamphetamine (Alltech, Barcelona, Spain); (Basf, Ludwigshafen, Germany), (Boehringer Ingelheim, Ingelheim, Germany); (Bristol-Myers Company, Nova York, USA); methylenedioxyamphetamine (MDA), methylenedioxyethylamphetamine (MDEA), methylenedioxymethamphetamine (MDMA) and methylenedioxymethamphetamine d5 (MDMA d5) (Cerilliant, Texas, USA); , ethylamphetamine and (Doping Analytical Section, Rigshospitalet, Copenhague); (European Pharmacopeia, Strasbourg, France); norephedrine (Impex-quimica, Barcelona, Spain); (Knoll, Ludwigshafen, Germany); (Laboratorio Llorente, Madrid, Spain); (LGC Standards, Luckenwalde; Germany); norpseudoephedrine (Mack GmbH , Illertissen, Germany); fencamfamine (Merck, Darmstadt, Germany); etaphedrine (Merrel Dow, Horgen, Switzerland); (Ministerio de Sanidad y Consumo, Madrid, Spain); crotetamide and carphedon (National Analytical Refrence Laboratory, Pymble, Australia); p-hydroxy-amphetamine, , , p-methylamphetamine, , , , cropropamide, and fenetylline (NMI Australian Government, Sidney, Australia); (Orion-Farmos Pharmaceuticals, Espoo, Finland); (Pfizer, Milan, Italy); (Roche, Monza, Italy); , , , methylephedrine, (d-), , methylhexaneamine, , , , , , diphenylamine used as internal standard (ISTD), chloramphetamine, , , , , pentetrazole and (Sigma-Aldrich, St Loiuse, MO, USA); p-hydroxy-metamphetamine, and pipradol (Toronto Research Chemicals, Toronto, Canada); (Uriach, Barcelona, Spain); , , , , 1,4- dimethylpenthylamine, 2-amino-N-ethylphenylamine and (World Association of Anti-Doping Scientists). Acetonitrile (LC gradient grade) was purchased from Merck (Darmstadt, Germany), formic acid (LC/MS grade) and MBTFA (N-methyl-bis- trifluoroacetamide) were purchased from Sigma-Aldrich (St Loiuse, MO, USA). All other solvents and reagents were of analytical grade. Ultrapure water was obteined using a Milli-Q purification system (Millipore Ibérica, Barcelona, Spain).

Standard solutions

Stock standard solutions of 1 mg/mL of the compounds in study were prepared in methanol. Working solutions of 100 and 10 µg/mL were prepared as 1:10 and 1:100 dilutions of the stock solution. All solutions were stored at −20 ºC.

Sample preparation procedures

Preparation for LC-MS/MS

Urine sample (0.5 mL) was centrifuged at 3500 rpm for 5 min. An aliquot of 300 µL was transfered to vial and diluted 1:5 with a mixture of ultrapure water:acetonitrile (99:1, v/v) containing formic acid (1%) and diphenylamine (1 μg/mL) as internal standard (ISTD).

Preparation for GC/MS (underivatized extracts)

A previously described procedure was used[30]. Urine samples (5 mL) were spiked with 25 µL of the ISTD solution (diphenylamine, 1 μg/mL) and were made alkaline with 0.5 mL of 5M KOH. Samples were extracted with tert- butylmethyl eter (2 mL) using salting-out (anhydrous sodium sulphate, 3 g). After agitation (40 mov/min, 20 min) and centrifugation, the organic phase was evaporated to dryness under nitrogen stream in a water bath at 25ºC. The residue was reconstituted with methanol (100 µL) and analyzed by GC/MS.

Preparation for GC/MS (derivatized extracts)

Samples were extracted as indicated above using MDMA D , as ISTD (25 μL of 5 a 100 μg/mL solution). The organic phase was evaporated to dryness, reconstituted with 100 µL of MBTFA and derivatized at 60ºC for 30 min in a dry bath. Extracts were analyzed by GC/MS.

UHPLC-MS/MS instrumental conditions

LC-MS/MS analyses were carried out using a triple quadrupole mass spectrometer (Xevo TQ MS) provided with an orthogonal Z-spray-electrospray interface (ESI) (Waters Associates, Milford, MA, USA) coupled to an ultraperformance liquid chromatographic (UPLC) system (Acquity, Waters Associates) for the chromatographic separation. Cone gas as well as desolvation gas was nitrogen. The desolvation gas flow was set to approximately 1200 L/h and the cone gas flow to 50 L/h. The nitrogen desolvation temperature was set to 450ºC and the source temperature to 150ºC. Argon was used as collision gas. Electrospray in positive ionization mode was used and the capillary voltages was set at 3.5 kV.

Detection was performed in multiple reaction monitoring mode (MRM). Electrospray ionization (ESI) working parameters (cone voltage, collision energy) were optimized for each analyte by direct infusion of individual standard solutions (10 µg/mL) into the mass spectrometer at a flow rate of 10 µL/min with mobile phase (50:50,v:v, water:acetonitrile with 0.01% formic acid) at 200 µL/min. Cone voltage, ion transitions and collision energies used for each analyte are listed in Table 1. Interscan times of 3 ms, interchannel delays of 3 ms and dwells time between 5 to 160 ms were applied. Acquisition was optimized to 9-12 data points per peak. MassLynx 4.1 software was used to acquire and process data (Waters Corporation, Milford, MA).

The chromatographic separation was carried out on a Waters Acquity UPLCTM system using an Acquity BEH C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size) (Waters Corporation, Milford, MA). The column temperature was set to 45 ºC. The mobile phase consisted of deionized water with 0.01% formic acid (A) and acetonitrile with 0.01% formic acid (B). Separation was performed at a flow-rate of 0.6 mL/min with the following gradient conditions: from 0 to 1.2 min, 1% B, increase to 90% B in 2.6 min, 90% B during 0.2 min, decrease to 1% B in 0.1 min and equilibration to initial conditions for 0.90 min. The total run time was 5 min and the sample volume injected was 10 μL.

For the separation of isomeric compounds (Groups A-F, Table 3), four different optimized gradients were used: Gradient A/B: 0 min 1% B, increase to 3% B in 4.2 min, increase to 90% B in 3.8 min, 90% B for 0.5 min, decrease to 1% B in 0.1 min, and equilibration for 1.4 min. Gradient C: 1% B for 1min, increase to 15% B in 7 min, increase to 90% B in 1 min, 90% B for 1 min, decrease to 1% B in 0.1 min, and equilibration for 1.9 min. Gradient D: 5% B for 1.2 min, increase to 90% B in 16.8 min, 90% B for 0.7 min, decrease to 5% B in 0.1 min, and equilibration for 1.2 min. Gradient E/F: 1% B for 1.2 min, increase to 90% B in 6.8 min, 90%B for 0.7 min, decrease to 1%B in 0.1 min, and equilibration for 1.2 min.

GC/MS instrumental conditions

GC/MS analysis were conducted using a 5890 GC gas chromatograph coupled with a 5973 MS mass spectrometer (Agilent Technologies, California, USA). The instrument was equipped with an autosampler 7673 (Agilent Technologies). The capillary column used was a 5% phenylmethyl silicone (12m length, 0.2mm internal diameter, 0.33µm film thickness) (Agilent Technologies). The carrier gas was helium (flow rate 0.9-1.1 mL/min). The injector and the transfer line temperatures were 280°C. The volume of sample injected was 3 µL in split mode (1:10-1:15).

Different temperature programs were applied to achieve the separation of the isomeric compounds: Ramp 1: initial temperature was kept at 110°C during 14 min, increasing to 280ºC at 20°C/min and held at 280ºC for 4 min. Ramp 2: initial temperature was set at 90°C, increasing to 300ºC at 20°C/min and held at 300ºC for 4 min. Ramp 3: initial temperature was set at 90°C, increasing to 290ºC at 20°C/min and held at 290ºC for 5 min. The analyses were performed in scan mode (m/z 50 to 600 and 40-400) and the characteristic ions for each compounds are listed in Table 3.

Validation study

The method was validated for qualitative purposes according to a previously described protocol[31].

Selectivity and specificity were studied by the analysis of 50 different blank urine samples. The absence of any interfering substance at the retention time of the compounds of interest and the internal standard was verified.

The detectability of the analytes was evaluated by analysis of six replicates of blank urine samples spiked with the analytes at 50 ng/mL, corresponding to 50% of the minimum required performance limits (MRPL) defined by WADA[29].

Intra-assay precision was assessed by analysis of six replicates of blank urine samples spiked at two different concentrations 50 and 100 ng/mL (low and high quality control samples, respectively) on the same day. Intermediate precision was estimated by analysis of one replicate of a quality control sample spiked with a selection of compounds, on six different days. The precision was expressed as the relative standard deviation (RSD) of the ratio between the areas of the compound and the ISTD.

Matrix effect was evaluated by the analysis of six different urines spiked at a concentration of 50 ng/mL of each compound. The areas of the analytes in these samples were compared with the area obtained after the analysis of water spiked at the same concentration.

The carry over between samples was assessed by analysis of a blank sample just after analysis of a sample spiked with the analytes at high concentration.

Analysis of authentic urine samples

The screening method was applied to urine samples obtained in excretion studies involving the administration of single oral doses of the compounds to healthy volunteers. The clinical protocol was approved by the local Ethical Committee (CEIC-IMAS, Barcelona, Spain). Urine samples were collected before administration and up to 24 h after administration at different collection periods. The following compounds were studied: amphetamine, benfluorex, clobenzorex, ephedrine, etaphedrine, fencanfamine, fenfluramine, fenproporex, heptaminol, isometheptene, MDMA, mephedrone, mefenorex, methamphetamine, methylhexaneamine, methoxyphenamine, niketamide, phentermine, pipradol, prolintane and pseudoephedrine.

The method was also applied to routine anti-doping control for a period of one year with analysis of more than 3000 samples RESULTS AND DISCUSSION

Method development

The compounds studied are nitrogen containing compounds and most of them bear a phenylalkylamine structure (Table 1). For that reason, they were easily ionized in positive mode ESI, specially using the mobile phase of water and acetonitrile slightly acidified with formic acid. Most of the compounds formed the protonated molecule [M+H]+. In source fragmentation was observed for pipradol, where the ion resulting from dehydratation of the protonated molecule, + [M+H-H2O] , was the most abundant specie (Table 1).

ESI working parameters are listed in Table 1. In general, low cone voltages and collision energies were used due to the molecular size of the compounds and the stability of the product ions formed. Due to similarities in structures among the compounds of the group, similar fragmentation routes and product ions were also obtained. Different isomeric groups are observed, and they are indicated in Table 1. The product ion mass spectra of the compounds included in each isomeric group are presented in Figure 1. Data acquisition for analysis of urine samples was performed in MRM mode. For screening purposes, one ion transition was used to monitor most of the compounds. However, for some compounds two ion transitions were measured to increase specificity. A total of 72 ion transitions were measured with time windows of 0.3-0.6 min around the expected retention time of the compound (Table 1). The dwell times were automatically adjusted by the instrument software to obtain between 9 and 12 data points per peak, allowing an adequate definition of the chromatographic peaks.

Regarding chromatographic conditions, apart from formic acid, no other mobile phase additives were needed either to promote ionization or to improve chromatographic behavior of the studied compounds. The gradient was optimized to provide a reasonable short chromatographic time allowing for the analysis 63 compounds in only 5 min (Table 1). Taking into account the sample preparation used, the chromatographic gradient was optimized to elute polar matrix compounds (e.g. salts) before elution of the analytes in order to avoid problems due to matrix effects. For that reason, the initial percentage of organic solvent was very low, 1%. For the same reason, although not needed for chromatographic separation, the final content of the organic solvent was increased to 90% in order to have a good clean-up of the chromatographic column after each sample analysis.

Due to the high ionization efficiency obtained for the majority of compounds, a simple dilution of the urine was studied as a sample preparation. Due to the complexity of the urine matrix, it is expected that urine dilution results in significant matrix effects produced by urinary interfering compounds that co- elute with the analytes and interfere with ionization. A dilution factor of 5 was selected as a compromise between sensitivity and matrix effects. The composition of the solvent used for dilution was optimized to obtain adequate signal intensities and chromatographic peak shapes for all compounds. Splitted peaks were obtained for some analytes due to the pH environment and the fact that the are in equilibrium with their respective conjugated acids. In order to avoid them, the diluting solution was acidified to favor one of the species. Trifluoroacetic acid (TFA) and formic acid were evaluated, and results obtained were similar for both modifiers. Taking into account that TFA may produce problems of contamination in the instrument, formic acid was selected. Different percentages of formic acid in the dilution solvent were studied (0.1%, 1% and 5%). Improvement of peak shape was observed as the percentage of acid increased, especially for some analytes. However, high percentages of acid may also result in contamination problems in the instrument. Therefore, 1% formic acid was selected as a compromise. Finally, the percentage of organic modifier in the dilution solvent was also studied. A mixture containing the same proportion of water and acetonitrile as the initial composition of the mobile phase (99:1,v/v) was selected, that offered optima signals and peak shapes, especially for some compounds (e.g., p-hydroxy-amphetamine).

Validation of the screening method The screening method was validated for qualitative purposes for the majority of compounds. A summary of the validation results is presented in Table 2.

Selectivity and specificity were evaluated by analysis of fifty blank urine samples. The method showed good selectivity and specificity with no interfering peaks. Some interfering peaks were observed close to the expected retention time of some analytes, and two characteristic ion transitions were monitored to improve selectivity in these cases (see Table 1). Some isomeric compounds were not chromatographically separated (Table 1, Figure 1) and unequivocal identification was not possible in the screening step. The identification of isomeric compounds is described in the specific chapter.

The fulfillment of the WADA criteria regarding the detectability of the analytes was verified by analysis of samples spiked at 50% of the MRPL[29]. For all analytes, a signal/noise ratio greater than 3 was obtained, indicating that the limit of detection is lower than 50 ng/mL for all analytes. .

Moreover, the detectability was evaluated using six different urine samples to study the matrix effect. Matrix effect was observed for most of the compounds, either decreasing or increasing the response, and ranged from 60 to 255% with RSD lower than 35% for the majority of compounds, indicating reproducibility between urine samples (Table 2). The presence of matrix effect was expected, taking into consideration the simple sample preparation and the fast chromatographic analysis. It is accepted that the matrix effect in qualitative analysis is less critical mainly for analytes showing high ionization efficiency, as it is the case of stimulants, and the matrix effect is evaluated to ensure that ion suppression does not make the analyte undetectable at the required concentration level in a specific urine [29]. Despite the matrix effect observed in our method, the signal/noise ratio of the analytes spiked at 50 ng/mL was in all samples tested greater than 3, allowing a correct detection of all substances at the MRPL required by WADA and demonstrating the suitability of the method.

Intra-assay precision (n=6) was calculated by analysis of quality control samples at two concentrations levels, 50 and 100 ng/mL (Table 2). Appropriate repeatability was obtained, with RSD values within the range 0.7-27.1% and 0.3-23.1% for the low and high quality control samples, respectively. Regarding intermediate precision, estimated for a selection of analytes during routine work, higher RSDs were obtained. These high RSDs obtained for some analytes are probably due to differences between days in the daily status of the mass spectrometer and of the chromatographic system. Small differences in chromatographic analysis can produce distinct elution of matrix interferences and, in consequence, in matrix effects on the analytes. So, taking into account that the methodology proposed is used as screening tool and all the analytes are detected at the level required the high values of intermediate precision are not considered a drawback. Carry over between samples was also studied. No signal was observed in blank samples analyzed after samples spiked with high concentrations of the compounds.

As a final validation, administration study samples were analysed. The method was able to detect the administration of amphetamine, benfluorex, clobenzorex, etaphedrine, fencanfamine, fenfluramine, fenproporex, heptaminol, isometheptene, MDMA, mephedrone, mefenorex, methamphetamine, methylhexaneamine, methoxyphenamine, niketamide, phentermine, pipradol, prolintane, ephedrine and pseudoephedrine in samples collected up to 24h after intake of a single oral dose. As examples in Figure 2, chromatograms of blank urine samples are compared with those obtained for samples collected after administration of clobenzorex, fenfluramine, heptaminol, niketamide and benfluorex. For some of the compounds, the monitoring of metabolites in addition to the unchanged helps in the detection of positive urines. As can be seen in Figure 2, after administration of clobenzorex, peaks of the parent and the metabolites, p-hydroxy-amphetamine and amphetamine, are observed in urine. For fenfluramine, the metabolite norfenfluramine is also detected. In these regards, the future incorporation of phase II metabolites (glucuronides or sulphates), not limited by the sample preparation step, may considerable improve the detection of some prohibited stimulants[27,32]

In order to evaluate the reliability for routine work, the described method was applied to the analysis of authentic doping control routine samples received in the laboratory for more than one year. In our hands, the system demonstrated to be stable and reliable with simple maintenance operations. Moreover, the method was subjected to the external quality control of WADA for a period of one year with successful results in the identification of positive samples to prolintane, isometheptene, cyclazodone, norpseudoephedrine, ephedrine and pseudoephedrine.

Identification of isomeric stimulants

As mentioned before, there were some isomeric groups including compounds with the same ion transitions and very close retention time in the conditions of the screening analysis (Table 1). These isomeric groups are indicated as A, B, C, D, E and F in Table 1, and their structures and product ion mass spectra can be seen in Figure 1. Alternative approaches have to be used for identification of a presumptive analytical finding. The procedures available to identify isomeric compounds are listed in Table 3.

The first possibility is the reanalysis of the samples using LC specific gradient for the separation of each isomeric group, and specific MS/MS methods including characteristic ion transitions (Table 3). In this study, compounds within all isomeric groups were separated using improved chromatographic gradients, and the separations achieved are shown in Figure 3. As an example, for the identification of dimethylamphetamine, ethylamphetamine and mephentermine (group F), in addition to the use of a specific gradient elution (gradient F), the presence of the product ion at m/z 133 in the mass spectra of the precursor m/z 163 helps in the identification of the compound as mephentermine, as the product ion is not present in the mass spectra of the other two compounds (see Figures 1 and 3).

Another possibility to identify isomeric compounds is the use of GC-MS analysis, without or with derivatization (Table 3). In that case, the urine sample has to be subjected to an extraction procedure before the chromatographic analysis (see experimental section). Using GC-MS without derivatization, identification of different isomeric groups can be easily achieved because of the different retention times and also different electron ionization mass spectra (see groups C and E). The separation of the diastereomeric pairs norephedrine/norpseudoephedrine (group A) and ephedrine/pseudoephedrine (group B) can be accomplished by GC/MS using a very slow temperature program (ramp 1).

Using GC-MS as TFA-derivatives, some isomeric compounds can be also distinguished because of the different retention times and electron ionization mass spectra (see groups C, E and F)[33].

The method developed, not being a chiral oriented analysis, does not separate the d- and l-enantiomers of amphetamine and metamphetamine. For these enantiomeric compounds (group C), a chiral separation, such as the diastereomeric derivatization with l-N- trifluoroacetyl-1-prolyl chloride[34] should be carried out.

CONCLUSIONS

A fast screening method based on UHPLC-MS/MS analysis was developed and validated for the simultaneous detection of 63 stimulants in urine at the concentration levels required by WADA. Neither extraction nor evaporation is required for processing the urine sample, which minimizes the loss of volatile analytes during the sample preparation.

The existence of isomeric stimulants with close retention times and/or similar ion transitions did not allow the unequivocal identification of these compounds in the initial testing procedure. Different possibilities for separation and identification of isomeric compounds were proposed, including the use of improved chromatographic gradients or the analysis by GC-MS without or with derivatization.

As a result of simple sample preparation and short LC-MS/MS analysis, a fast turn-around time is achieved. These features make the developed method highly interesting for routine anti-doping purposes. ACKNOWLEDGEMENTS

The financial support received from Consell Català de l’Esport and DIUE (2014 SGR 692) (Generalitat de Catalunya; Spain) is acknowledged. Technical assistance of Noemi Haro is gratefully acknowledged.

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[34] S.M. Wang, T.C. Wang, Y.S. Giang. Simultaneous determination of amphetamine and methamphetamine enantiomers in urine by simultaneous liquid- liquid extraction and diastereomeric derivatization followed by gas chromatographic-isotope dilution mass spectrometry. J. Chromatogr B, 2005, 816, 131. Table 1. Compounds included in the screening procedure: monoisotopic mass (MM), retention times (RT), relative retention times (RRT), precursor ion (PI), product ion (DI), cone voltage (CV) and collision energy (CE). Isomeric group indicates those compounds with similar retention time and product ion mass spectra.

MM RT PI DI CV CE Isomeric Analyte RRT (Da) (min) (m/z) (m/z) (V) (eV) group p-hydroxy-amphetamine 151.10 1.82 0.49 152 135 15 10 - p-hydroxy-metamphetamine 165.12 1.94 0.53 166 135/107 20 10/15 - Heptaminol 145.15 1.99 0.54 146 128 15 10 - Benzylpiperazine 176.13 2.03 0.55 177 91 25 20 - Norpseudoephedrine 151.11 2.04 0.55 152 134 10 5 A Norephedrine 151.11 2.04 0.55 152 134 10 5 A Amiphenazole 191.05 2.10 0.57 192 117/106 30 25 - Ephedrine 165.12 2.14 0.58 166 148 10 10 B Pseudoephedrine 165.12 2.15 0.58 166 148 10 10 B Methylephedrine 179.13 2.19 0.59 180 162 30 10 - Metamphetamine (d/l) 149.12 2.21 0.60 150 119/91 20 10/15 C Phenprometamine 149.12 2.21 0.60 150 119/91 20 10/15 C Phenmetrazine 177.12 2.22 0.60 178 115 30 35 - Amphetamine 135.10 2.23 0.60 136 119/91 15 10/15 - Etaphedrine 193.15 2.23 0.60 194 176/60 25 15/20 - MDA 179.09 2.24 0.61 180 163 15 10 - Phendimetrazine 191.13 2.25 0.61 192 146/117 32 25 - Caffeine 194.08 2.27 0.62 195 138 35 25 - Amfepramone 205.15 2.28 0.62 206 105 30 20 - Methylhexaneamine 115.14 2.29 0.62 116 57 15 10 D Phentermine 149.12 2.29 0.62 150 133/105 15 10/15 E MDMA 193.11 2.29 0.62 194 163 20 15 - Dobutamine 301.17 2.30 0.62 302 107 30 20 - Fenproporex 188.13 2.31 0.63 189 119 15 10 - Strychnine 334.17 2.31 0.63 335 184 35 40 - Mephentermine 163.14 2.32 0.63 164 133/91 22 15 F Dimethylamphetamine 163.14 2.32 0.63 164 119/91 22 15 F Tuaminoheptane 115.14 2.34 0.63 116 57 15 10 D Pentetrazole 138.09 2.35 0.64 139 96/69 25 15 - Ethylamphetamine 163.14 2.36 0.64 164 119/91 20 15 F Methoxyphenamine 179.13 2.36 0.64 180 149 20 15 - MDEA 207.13 2.36 0.64 208 163 20 15 - Fencamine 384.23 2.36 0.64 385 236 35 20 - p-methylamphetamine 149.12 2.37 0.64 150 133/105 15 10 E Ortetamine 149.12 2.39 0.65 150 133/105 10 10/15 E 1,4-dimethylpenthylamine 115.13 2.39 0.65 116 57 15 10 D Mephedrone 177.11 2.39 0.65 178 145 25 20 - Isometheptene 141.15 2.41 0.65 142 69 20 15 - Nikethamide 178.11 2.42 0.66 179 108 20 20 - Selegiline 187.14 2.43 0.66 188 91 20 15 - Fenetylline 341.19 2.43 0.66 342 207 30 25 - 2-amino-N-ethylphenylamine 177.15 2.43 0.66 178 133 20 15 - Chloramphetamine 169.07 2.47 0.67 170 153 15 10 - Norfenfluramine 203.09 2.52 0.68 204 187 20 10 - Furfenorex 229.16 2.53 0.69 230 148/81 20 15 - Chlorphentermine 183.08 2.54 0.69 184 167/125 15 10/15 - Carphedon 218.11 2.54 0.69 219 202/174 15 10/15 - Mefenorex 211.11 2.56 0.69 212 119 22 15 - Propylhexedrine 155.17 2.59 0.70 156 69 20 15 - Pipradol* 267.16 2.61 0.71 250 130 40 30 - Fencamfamine 215.17 2.65 0.72 216 171 20 15 - Benzphetamine 239.17 2.66 0.72 240 119/91 25 15/20 - Prolintane 217.18 2.68 0.73 218 105/91 30 20 - Fenfluramine 231.12 2.68 0.73 232 159 25 20 - Cyclazodone 216.09 2.72 0.74 217 146 20 15 - Clobenzorex 259.11 2.72 0.74 260 91 25 15 - Crotetamide 226.17 2.74 0.74 227 182/86 5 5 - Cropropamide 240.18 2.95 0.80 241 196 10 5 - Sibutramine 279.18 2.96 0.80 280 139/125 25 15 - Benfluorex 351.14 2.97 0.80 352 230 25 20 - Prenylamine 329.21 3.04 0.82 330 212 30 20 - Famprofazone 377.25 3.07 0.83 378 229 30 20 - Fenbutrazate 367.21 3.08 0.83 368 204 30 20 - ISTD 169.09 3.69 1.00 170 93 35 25 - *The precursor ion for pipradol is [M+H-H2O]+.

Table 2. Validation results: intraday and intermediate precisions, and matrix effect.

Intraday precisions (n= 6) Analyte Intermediate precision (n=6) Matrix effect Conc (50 ng/mL) Conc (100 ng/mL) RSD (%) RSD (%) Conc (ng/mL) RSD (%) mean (%) RSD(%) Amfepramone 4.0 5.6 100 36.8 - - Amiphenazole 6.0 2.9 100 31.8 71.3 56.7 Amphetamine 5.6 3.7 100 53.5 - - Benfluorex 0.7 0.3 100 10.2 122.2 28.8 Benzphetamine 2.5 0.5 100 6.5 106.7 29.3 Benzylpiperazine 3.5 4.8 100 24.0 - - Caffeine 1.4 11.7 100 11.3 - - Carphedon 26.0 8.7 100 34.3 254.9 26.4 Chloramphetamine 10.3 9.7 - - - - Chlorphentermine 9.7 21.9 - - 110.9 30.4 Clobenzorex 5.4 5.8 100 49.2 107.1 31.4 Cropropamide 6.1 10.2 100 48.6 107.2 24.0 Crotetamide 2.9 4.7 100 31.8 112.9 6.8 Cyclazodone 25.0 1.9 100 9.5 181.7 48.3 Dimethylamphetamine 13.4 6.6 - - - - Dobutamine 16.1 7.6 100 39.0 111.0 31.7 Ephedrine 14.0 8.0 1000 31.7 99.9 20.7 Etaphedrine 6.1 2.3 100 69.4 97.3 26.0 Ethylamphetamine 12.2 11.6 100 8.3 126.5 32.9 Famprofazone 3.0 8.5 - - 99.8 9.5 Fenbutrazate 5.1 4.2 100 12.1 119.3 38.3 10.6 4.5 100 12.1 - - Fencamine 15.6 22.6 100 78.5 87.6 28.3 Fenetylline 12.8 10.9 100 49.8 - - Fenfluramine 11.8 4.4 100 16.4 118.4 8.4 Fenproporex 13.6 7.4 100 59.6 100.1 35.6 Furfenorex 22.7 7.6 100 64.1 118.7 40.2 Heptaminol 13.1 7.7 - - - - Isometheptene 4.6 1.2 100 10.7 97.7 12.0 MDA 1.0 5.2 - - - - MDEA 20.7 4.3 100 45.8 102.3 17.4 MDMA 14.4 7.2 100 38.5 119.6 17.4 Mefenorex 17.2 3.8 100 21.6 117.3 18.9 Mephentermine 10.4 6.2 100 11.1 - - Methamphetamine(d-) 3.7 3.5 100 42.0 94.1 13.1 Methamphetamine(l-) 13.6 7.1 - - - - Methoxyphenamine 7.0 6.3 100 55.0 - - Methylephedrine 8.8 7.1 1000 58.5 118.6 22.9 Methylhexaneamine 7.8 6.2 100 40.0 128.2 22.5 Nikethamide 17.7 2.6 100 21.0 90.0 17.4 Norfenfluramine 10.3 6.2 100 66.1 72.2 33.0 Norpseudoephedrine 2.2 5.5 1000 59.0 127.6 21.4 Norephedrine 9.2 19.6 - - - - Pentetrazole 1.3 1.3 - - - - Phendimetrazine 4.2 6.0 - - - - Phenmetrazine 10.8 9.9 100 9.2 99.1 12.7 Phenpromethamine 2.9 6.3 - - - - Ortetamine 6.0 4.0 - - - - Phentermine 13.9 6.1 100 61.2 59.5 44.6 Pipradol 4.0 6.1 100 23.8 - - p-Methylamphetamine 18.0 15.6 100 51.8 - - p-hydroxy-amphetamine 27.1 6.3 100 68.8 82.5 19.8 p-hydroxy-metamphetamine 16.6 3.8 100 - 149.0 13.9 Prenylamine 2.1 1.0 100 17.6 185.9 21.9 Prolintane 13.3 5.6 100 10.4 109.6 6.5 Propylhexedrine 3.9 4.6 100 52.4 89.8 25.2 Pseudoephedrine 4.7 7.2 - - - - Selegiline 3.0 4.0 - - - - Sibutramine 5.1 3.4 100 7.5 - - Strychnine 13.8 22.1 100 15.2 - - Tuaminoheptane 6.4 15.4 100 32.3 78.3 39.2 1,4-dimethylpenthylamine 17.7 23.1 - - - -

Table 3. Separation conditions by LC-MS/MS and GC-MS for the identification of the compounds of the diferent isomeric groups. For each condition, retention time (RT) and characteristic ion transitions or m/z of the analytes.

GC/MS GC/MS LC-MS/MS Group Identification of isomers Compound underivatized N-TFAderivatives RT RT RT Transitions m/z m/z (min) (min) (min) Norpseudoephedrine 2.57 152>134;117 3.52 44,77,105 - - LC-MS/MS (Gradient A/B) Norephedrine 2.24 152>134;117 3.59 44,77,105 - - A/B GC-MS underivatized (Ramp 1) Ephedrine 3.30 166>148;133 4.56 58,77,105 - - Pseudoephedrine 3.70 166>148;133 4.66 58,77,105 - - LC-MS/MS (Gradient C) GC-MS underivatized (Ramp 2) C Metamphetamine(d) 4.56 150>119;91 1.82 58,91,134 2.98 91,110,118,154 GC-MS-N-TFA derivatives (Ramp 3) Metamphetamine (l) 4.56 150>119;91 1.82 58,91,134 2.97 91,110,118,154 GC-MS chiral separationa Phenprometamine 4.56 150>119;91 1.86 44,77,91 2.91 105,118,140 Methylhexaneamine 2.76 116>57;43 n.ab n.a n.a n.a D LC-MS/MS (Gradient D) Tuaminoheptane 3.14 116>57;43 n.a. n.a. n.a. n.a 1,4-dimethylpenthylamine 2.94 116>57;43 n.a. n.a. n.a n.a LC-MS/MS (Gradient E) Phentermine 2.86 150>133;105 1.72 58,91,134 2.42 91,114,132,154 E GC-MS underivatized (Ramp 2) p-methylamphetamine 3.09 150>133;105 2.08 44,77,91,105 2.85 105,132,140 GC-MS N-TFA derivatives (Ramp 3) Ortetamine 3.00 150>133;105 2.12 44,77,91,105 2.88 105,132,140 LC-MS/MS (Gradient F) Dimethylamphetamine 2.80 164>119;91 2.19 72,91,148 2.30c 72,91 F GC-MS underivatized (Ramp 2) Ethylamphetamine 2.92 164>119;91 2.11 44,72,91,148 3.22 91,118,140,168 GC-MS N-TFA derivatives (Ramp 3) Mephentermine 2.96 164>133;91 2.25 72,91,148 3.15d 91,110,168 Diphenylamine (Gradient A/B) 7.38 170>93 - - - - Diphenylamine (Gradient C) 9.31 170>93 - - - - Diphenylamine (Gradient D) 10.11 170>93 - - - - ISTD Diphenylamine (Gradient E/F) 5.99 170>93 - - - - Diphenylamine (Ramp 1) - - 15.80 169 - - Diphenylamine (Ramp 2) - - 4.46 169 - - MDMA D5 (Ramp 3) - - - - 4.95 158

a b c d Chiral separation of d- and l-metamphetamine (see reference 34), not analyzed, underivatized, partially derivatized. Most abundant ions are underlined.

Figure 1. Chemical structures, product ion mass spectra and fragmentation pattern of isomeric estimulants.

Figure 2. LC-MS/MS results of urine samples obtained after administration of clobenzorex, fenfluramine, heptaminol, nikethamide and benfluorex. Chomatograms of the characteristic ion trasitions of the analytes obtained after analysis of a blank sample (up), and after analysis of a post administration sample (down). Results of the different metabolites detected are shown for clobenzorex and fenfluramine (A, B).

Figure 3. Chromatograms using specific chromatographic gradients for the separation of isomeric compounds.

A. Norephedrine B. Ephedrine Norpseudoephedrine Pseudoephedrine OH OH 148 148 134 CH3 100 134 100 NH2 HN CH3 CH3

117 %% %% 115 91 117 133 56 56 70 91 166 0 152 0 50 100 150 50 100 150 C. Phenprometamine C. Metamphetamine (d/l)

CH3 NH NH CH CH 119 3 119 3 150 100 150 100 91 CH3 91 119 91 %% 119 %%

0 m/z 0 50 100 150 50 100 150 D.Methylhexaneamine D. Tuaminoheptane D. 1,4-dimethylpenthylamine NH2 99 43 CH NH2 H3C CH3 3 57 H3C 57 57 100 100 43 57 100 NH CH3 CH3 2 H3C 99 43 57 99 CH3 %% %%% %% 57 116 116 116 43 99 43 43 0 0 0 50 100 150 50 100 150 50 100 1500 E. Phentermine E. P-methylamphetamine E. Ortetamine 133 91 CH3 NH NH2 2 91 NH 105 105 100 2100 100 H3C CH3 CH CH3 3 H3C 105 105 133 133 %% %%% %% 133 133 133 105 150 150 150 0 0 0 m/z 50 100 150 50 100 150 50 100 150 F. Dimethylamphetamine F. Ethylamphetamine F. Mephentermine

CH3 N 133 91 CH3 91 NH CH3 91 100 100 100 91 91 NH 91 CH3 CH3 CH3 119 H3C CH3 119 %%

164 %% %% 119 133 119 164 164 46 46 105 0 0 0 50 100 150 50 100 150 50 100 150 A. Clobenzorex p-hydroxy-amphetamine Amphetamine

100 260>91100 152>135 100 136>91 2.42 103 3.75 103 2.21 105 % % %

0 Time 0 Time 0 Time 2.60 2.70 2.80 1.60 1.70 1.80 2.10 2.20 2.30

B. Fenfluramine Norfenfluramine

100 232>159 100 204>187 4.69 103 2.86 104 % %

0 Time0 Time 2.50 2.60 2.70 2.40 2.60

C. Heptaminol D. Nikethamide E. Benfluorex 100 146>128 100 179>108 100 352>230 9.38 103 6.76 103 1.85 103 % % %

0 Time 0 Time 0 Time 2.00 2.10 2.20 2.30 2.40 2.50 2.90 3.00 3.10 A. Clobenzorex p-hydroxy-amphetamine Amphetamine

100 100 100 260>91 152>135 136>91 9.60 106 5.32 105 8.98 107 % % %

0 Time 0 Time 0 Time 2.60 2.70 2.80 1.60 1.70 1.80 2.10 2.20 2.30

B. Fenfluramine Norfenfluramine

100 100 232>159 204>187 1.34 108 2.16 107 % %

0 Time 0 Time 2.50 2.60 2.70 2.40 2.60

C. Heptaminol D. Nikethamide E. Benfluorex

100 100 100 146>128 179>108 352>230 1.34 105 7.04 105 2.66 105 % % %

0 Time 0 Time 0 Time 2.00 2.10 2.20 2.30 2.40 2.50 2.90 3.00 3.10 A. Norephedrine 152>134 B. Pseudoephedrine 166>148 100 100 3.30 2.24 2.57 Ephedrine Norpseudoephedrine 3.70 %%% %%

0 Time 0 Time 2.00 3.00 4.00 2.00 3.00 4.00 C. Methylhexaneamine Phenprometamine 150>119 D. 116>57 4.46 2.76 2.94 100 4.56 100 Tuaminoheptane 3.14 Metamphetamine 1,4-dimethylpenthylamine % %%

0 Time 0 Time 3.00 4.00 5.00 2.00 3.00 4.00 E. Phentermine 3.00 150>133 100 2.86 3.09 p-methylamphetamine %%

Ortetamine 0 Time 2.00 3.00 4.00 F. 164>119 F. 164>133 Dimethylamphetamine 100 100 2.96 2.80 Ethylamphetamine Mephentermine

2.92 %% %

0 Time 0 Time 2.00 3.00 4.00 2.00 3.00 4.00